Food Chemistry: X 21 (2024) 101152

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Food Chemistry: X
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Amine vapor-responsive ratiometric sensing tag based on HPTS/TPB-PVA

fluorescent film for visual determination of fish freshness
Qian Han a, c, 1, Min Yang d, 1, Zexin Zhang d, Xinwen Bai d, Xiuying Liu a, c, *, Zhenhua Qin a, b, *,
Wei Zhang a, c, Pingping Wang a, c, Lijie Zhu a, c, *, Zaixi Shu a, c, Xuepeng Li d
a
School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, Hubei 430028, China
b
School of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan, Hubei 430028, China
c
Key Laboratory for Deep Processing of Major Grain and Oil, Ministry of Education Wuhan, Hubei 430028, China
d
College of Food Science and Technology, Bohai University, Jinzhou, Liaoning 121013, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, amine vapor-sensitive films with ratiometric fluorescence attributes were developed. The pH-
Fluorescent film sensitive fluorescein 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) and its tetraphenylethylene
Amine vapor derivative (TPB) were selected as ratiometric indicators and incorporated into a polyvinyl alcohol (PVA) matrix
Sensing tag
to produce HPTS/TPB-PVA films. The films responded well to amine vapors, and the interference of aromatic
Fish freshness
Ratiometric indicator
vapors did not substantially affect the fluorescence signals of the films. Under UV light at a wavelength of
365 nm, the fluorescence of the films changed from dark pink to light pink and finally to yellow when the
freshness of the fish was visually checked during storage. In addition, the color difference values of the films
showed a positive correlation with the total volatile basic nitrogen (TVB-N), ranging from 12.7 to 24.8 mg/100 g
at 25 ◦ C and 8.4 to 25.6 mg/100 g at 4 ◦ C, respectively. This indicates that fluorescent films have good potential
for quantifying fish freshness in the near future when connected to an automatic data processing system based on
color differences.

1. Introduction
Food spoilage results in food waste. Approximately one-third of the
global food supply, equivalent to 1.3 billion tons, is wasted annually (Ma
et al., 2021). Monitoring food quality in real-time is essential to satisfy
consumer expectations, reduce food waste, and ensure food safety. In
particular, aquatic products rich in proteins can easily degenerate dur­
ing transport and storage, producing harmful substances and negatively
impacting consumer health. (Duan, Li, Wang, Lin, Wang, 2023; Bondoc
& Șindilar, 2002; Bondoc, 2014). However, the evaluation of food
freshness relies mainly on the use of instruments. It is difficult for re­
tailers and consumers to identify whether food in packaging is fresh.
Therefore, developing a nondestructive, fast, and on-site evaluation
method will assist in addressing food waste issues.
Packaging is crucial in preventing food contamination and deter­
mining food grade (Hazarika, Konwar, Borah, Saikia, Barman, &
Hazarika, 2023). Recently, on-package sensing labels or films, a type of
intelligent packaging, has attracted increasing attention. These labels or
films offer information on food freshness and edible quality during the
entire storage process, showing color or visual signal changes in real
time and providing early warnings to consumers (You, Zhang, Wang,
Jin, Wei, & Wang, 2022). Among various indicators for developing
sensing labels or films, the use of pH indicators in food packaging has
been increasing (Amaregouda, Kamanna, & Gasti, 2022). In our previ­
ous study, we developed a series of colorimetric labels utilizing pH-
sensitive materials, such as bromocresol green pH indicator and poly­
aniline (Liu et al., 2020, Liu et al., 2020). In packaging, sensing labels
that use pH-sensitive dyes typically change color upon interaction with
volatile amines (Ezati, Bang, & Rhim, 2021). Consumers assess food
freshness based on the color of the sensing label. Nevertheless, there is a
need for further improvement in the sensitivity and stability of these
colorimetric labels.
The development of intelligent packaging shows considerable po­
tential with the use of fluorescent tags or films that exhibit exceptional
sensing capabilities, such as high sensitivity, rapid response, and
reversibility (Lai et al., 2021). Generally, fluorescence sensing films are

* Corresponding authors at: School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, Hubei 430028, China.
E-mail addresses: xiuyingliu727@126.com (X. Liu), qinzhenhua@whpu.edu.cn (Z. Qin), lijiezhu325@126.com (L. Zhu).
1
Qian Han and Min Yang contributed equally to this article.

https://doi.org/10.1016/j.fochx.2024.101152
Received 19 July 2023; Received in revised form 8 January 2024; Accepted 17 January 2024
Available online 20 January 2024
2590-1575/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Q. Han et al.
developed based on one type of luminogen, showing variations in
fluorescence intensity. However, in certain instances, only a slight
change in fluorescence intensity occurs, which is not expected for visual
detection (Jia et al., 2019). The sensitivity of single fluorescent signal
films used for detection with the naked eye requires further improve­
ment. Ratiometric fluorescent indicators, which integrate the signal
strengths of two signal units, can exhibit distinct fluorescent color
changes (Chen et al., 2022; Bigdeli et al., 2019; Gui et al., 2019). These
indicators have shown considerable potential for building sensing films
because the difference in fluorescent color is most noticeable to the
naked eye. Moreover, the ratiometric fluorescence technique can reduce
environmental interference and help obtain more accurate detection
results (Liu et al., 2021; Yao et al., 2013; Park, Kwon, Lee, Yoon, & Shin,
2020).
Therefore, in this study, we developed a proportional fluorescence
sensing label by combining a pH-sensitive indicator (HPTS) with a
reference indicator (TPB), using polyvinyl alcohol (PVA) as the matrix.
HPTS, which acts as a sensing indicator, was selected because of its
favorable amine-responsive behavior. TPB, a type of aggregation-
induced emission-active fluorogen (AIEgen), was selected for its
outstanding AIE attributes and high red fluorescence stability, serving as
an internal control indicator. The structural characteristics and
morphology of the indicator labels were evaluated using Fourier-
transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and
scanning electron microscopy (SEM). Additionally, we analyzed the
physical properties of the indicator labels, including the water vapor
transmittance, thickness, mechanical tensile strength, and solubility.
Finally, the feasibility of using sensing labels to monitor the freshness of
real samples was evaluated.
2. Materials and methods
2.1. Materials
Polyvinyl alcohol (PVA), HPTS fluorescein, and trimethylamine were
purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
(Shanghai, China). Ammonia was obtained from Tianjin Fuchen
Chemical Co. (Tianjin, China). TPB were purchased from Xuzhou Day­
ang Biochemical Technology Co., Ltd (Xuzhou, China). Octanal, ethyl
acetate, cyclohexanone and 2-methyl-furan were obtained from Maclin
Biochemical Technology Co. Ltd. (Shanghai, China).
Packaged fresh large yellow croakers were purchased from a local
market and transported to the lab within a period of 30 min. Before
testing, the innards, head, tail, and fins of the fish were removed.
2.2. Preparation of HPTS/TPB sensing films
The HPTS/TPB sensing film was produced using a solution casting
technique. To begin, 40 mL of deionized water was heated to 100 ◦ C.
Then, 2.0 g of PVA was dissolved in the heated water. After cooling to
25 ◦ C, a solution of glutaraldehyde solution (1 mL, 50 wt%) and HPTS
(5.0 mg) was added with magnetic stirring for 12 h. Afterward, the
above solution was added with TPB-tetrahydrofuran solution (4 mL,
1 mg/mL) to create the film casting solution. To eliminate any bubbles, a
petri dish with a diameter of 90 mm was coated with 40 mL of the film
casting solution and allowed to dry at 37 ◦ C for 10 h.
2.3. Characterization of HPTS/TPB sensing films
2.3.1. Film thickness
The films thickness (mm) was measured using a hand-held digital
micrometer (SL01-22, Biaokang, China), at 5 random locations on each
piece of the film.
2.3.2. Scanning electron microscope (SEM)
The surface and internal structure of the film samples were
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Food Chemistry: X 21 (2024) 101152
characterized using a SEM (SU8020, Hitachi, Japan). The observations
of the samples were performed under a 3.0 kV accelerating voltage.
2.3.3. Fourier transform infrared (FTIR) spectroscopy
FTIR spectra of the films were obtained using a FTIR spectrometer
(Scimitar 2000, Varian, USA). The film samples were scanned in the
range of 400 ~ 4000 cm− 1 with a resolution of 4 cm− 1. Each sample was
scanned 32 times.
2.3.4. X-ray diffractometer (XRD)
The changes in crystalline properties of the HPTS/TPB sensing films
were assessed using a XRD diffractometer (UItima IV, Rigaku, Japan).
The readings were conducted with 2θ ranging from 5◦ to 55◦ , operating
at 40 kV and 40 mA. The detection rate was set at 0.05◦ /s.
2.4. Physical properties of HPTS/TPB sensing films
2.4.1. Water solubility (WS)
The WS was tested using the method as reported (Li, Liu, Ye, Wang, &
Wang, 2015). The solubility of the sensing films was calculated using the
following equation:
m1 − m0
WS(%) = *100%
m
where “m0” represents the starting dry mass, and “m1” is the ending dry
mass.
2.4.2. Water vapor permeability (WVP)
The WVP was assessed following the American standard test methods
(ASTM E96M-05, 1995) with modification. Briefly, a testing beaker was
filled with 3 g of calcium chloride and sealed using the film sample. The
beaker was stored at 25 ◦ C (75 % RH), and then measured after 24 h.
The WVP was calculated using the following equation (Cao et al.,
2022):
WVP =
d × ΔM
S × ΔP
where ΔM is the mass difference of the beaker (g) after 24 h, d is the film
thickness (mm), S is the covered area of the film (m2), and ΔP (Pa) is the
partial pressure of vapor.
2.4.3. Water contact angel (WCA)
To determine the WCA, a droplet of 1 μL ultrapure water was
deposited onto the film surface, and measured using a WCA analyzer
(OCA25, Dataphysics, Germany) (Yang et al., 2023). The change in WCA
was recorded to analyze the surface hydrophobicity.
2.5. Functional properties of HPTS/TPB sensing films
The fluorescence response of the HPTS/TPB sensing films to amine
vapor, and vapor of aromatic chemicals was determined according to the
previous research (Liu et al., 2020, Liu et al., 2022). Briefly, the sensing
films were placed in the headspace of the 100 mL 0.01 % trimethyl­
amine, 0.01 % dimethylamine, and 0.01 % ammonia, respectively. Im­
ages of the sensing films under 365 nm ultraviolet light (UVSABIP, Puxi,
Beijing, China) were recorded using a smartphone every 30 min. For the
volatile aromatic vapor test, aromatic chemicals including 2-methyl-
furan, ethyl acetate, cyclohexanone, and octanal were selected and
tested using the same method.
2.6. Fish freshness evaluation during storage
2.6.1. Total viable counts (TVC)
TVC was measured following the Chinese standard GB 4789.2 with
some modification. Briefly, 10 g of sample and 90 mL of sterile
Q. Han et al.
physiological saline were placed in a sterile bag, and gently patted for
1 ~ 2 min to prepare a 1:10 diluted sample solution. The sample solution
was further diluted to 1:100, and 1 mL of the sample dilution was
injected into a sterilized agar plate, mixed with 15 ~ 20 mL of plate
counting agar medium. The plate was then incubated at 30 ◦ C, and the
number of colonies was record after 72 ± 3 h. The result was presented
as logarithm of colony forming units (CFU) per gram.
2.6.2. Total volatile basic nitrogen (TVB-N)
The determination of TVB-N value followed the method described in
GB 5009.228–2016 with slight modifications. In summary, 5 g of the
sample was mixed with 50 mL of deionized water and homogenized. The
homogenized sample was then filtered, and the filtrate was collected. In
the outer chamber of the diffusion dish, 1 mL of the filtrate and 1 mL of
saturated potassium carbonate solution were added. In the inner
chamber of the diffusion dish, 1 mL of 20 g/L boric acid solution and a
mixture of bromocresol green and methyl red indicators were added.
After sealing the diffusion dish, it was incubated at 37 ◦ C for 2 h. The
inner chamber filtrate was then titrated with 0.01 mol/mL hydrochloric
acid until a purple-red color was observed. The volume of hydrochloric
acid consumed during the titration was recorded, and the TVB-N content
was calculated.
2.6.3. Thiobarbituric acid (TBA)
The detection of TBA followed the method described in GB/T
35252–2017. In summary, 5 g of the sample was mixed with 50 mL of
7.5 % trichloroacetic acid, homogenized, and allowed to stand for
30 min. The mixture was then filtered, and the filtrate was collected.
Next, 5 mL of the filtrate was transferred into a test tube, and 5 mL of
0.02 mol/L 2-thiobarbituric acid solution was added. The mixture was
placed at 90 ◦ C for 30 min and then measured at 532 nm. The result of
the measurement was reported as mg malonaldehyde (MDA) per
kilogram.
2.6.4. pH measurement
According to the Chinese standard GB 5009.237, the pH values of
fish samples were determined. In general, 5 g of the sample was mixed
with 50 mL of a 0.1 mg/mL potassium chloride solution and homoge­
nized. The pH measurements were then carried out using a digital pH
meter. Three replicate measurements were performed.
2.7. Utilization of the films for visual assessing of fish freshness
Fish spoilage trials were performed at room temperature (25 ◦ C) and
chilled temperature (4 ◦ C). Briefly, 200 g of fresh large yellow croaker
samples were placed in PP food grade plastic boxes (20 × 10 × 5 cm).
Sensing films with a diameter of 1.5 cm were attached to the inside of
the top of each box. In order to facilitate interactions between the films
and vapors, four sticky spacers measuring 0.5 cm thick were placed at
the four corners of the tags, creating a gap with the cap. Throughout the
experiment, it was ensured that the plastic sample boxes remained
sealed. The fluorescence images of the sensing labels were captured by
exposing the sample box to 365 nm ultraviolet light. Under storage
conditions of 25 ◦ C, the images were recorded once every 4 h, while
under storage conditions of 4 ◦ C, the images were recorded once every
2 days. All experiments were conducted in triplicate.
2.8. Data analysis
The R, G, and B values were obtained by reading three spots located
on the upper, middle, and lower parts of each sensing label. The color
change sensitivity (S) was processed according to the following equation
(Huang, Zou, Zhao, Shi, Li, & Shen, 2015):
|R − R0 | + |G− G0 | + |B − B0
S(%) = × 100%
R+G+B
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Food Chemistry: X 21 (2024) 101152
where, R0 (red), G0 (green) and B0 (blue) were the starting values of the
images, and R, G, and B were the values at the sampling point.
The total color difference (ΔE) was determined according to the
following equation (Chen, Zhang, Bhandari & Yang, 2020):
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = (L − L0 )2 + (a − a0 )2 + (b − b0 )2
where, L0, a0 and b0 were the initial lightness, redness, and yellowness
values collected, and L, a, and b were the corresponding values at the
sampling point.
The color data, including R, G, B, L, a, and b values, were extracted
from the captured images using the Adobe Photoshop 2021 software.
The data were then processed using Origin 2018 software.
3. Results and discussion
3.1. Characterization of the HPTS/TPB-PVA sensing film
The HPTS/TPB-PVA film was developed based on the HPTS/TPB
ratiometric indicator; a schematic representation is shown in Scheme 1.
The red fluorescent reference dye TPB and indicator dye HPTS were
immobilized in PVA to prepare the indicator films. The sensing film
initially exhibited pink fluorescence and then interacted with the vola­
tile amines released from the sample, causing a change in the fluores­
cence signal of the sensing dye, TPB. This ultimately resulted in a shift in
the fluorescence color of the sensing film from pink to yellow.
SEM images of the PVA and HPTS/TPB-PVA films are shown in
Fig. 1. The PVA film exhibits a smooth, dense, and even surface. After
the addition of HPTS/TPB, small raised particles were observed owing to
HPTS/TPB aggregates. Thus, it can be concluded that the embedding of
HPTS/TPB did not have a notable impact on the film surface. However,
the cross-section of the HPTS/TPB PVA films was rougher than that of
the PVA film. This phenomenon can be explained by the interaction
between the HPTS/TPB and the PVA matrix. Compared to the pure PVA
film, the visual aspect of the HPTS/TPB-PVA film presented a slightly
pinkish color. (Fig. 1 E). The thickness of different batches of HPTS/TPB
sensing films was measured, and results ranged between 0.183 ± 0.018
to 0.247 ± 0.015 mm.
Fig. 1 F shows the FTIR spectra of the PVA and HPTS/TPB PVA films.
The peaks at approximately 3353, 2949, 1739, and 1096 cm− 1 indicate
–OH, –CH, H–O–H stretching, and C–O stretching, respectively (Cao
et al., 2022; Zhou et al., 2023). These four peaks were observed in both
spectra. After embedding HPTS/TPB, the C–O peak shifted from 1096
Scheme 1. Schematic representation of amine response of the film.
Q. Han et al.
Fig. 1. SEM images of surfaces and cross-sections of (A, B) PVA film, and (C, D) HPTS/TPB-PVA film; (E) visual appearance of the films; (F) FTIR curves of pure PVA
and HPTS/TPB-PVA film; (G) XRD curves of pure PVA and HPTS/TPB PVA film.
to 1083 cm− 1, representing changes in C–O stretching. The peaks at
1733 (H–O–H stretching) and 1083 cm− 1 (C–O stretching) of the
HPTS/TPB-PVA spectra were sharper, verifying the interaction between
HPTS/TPB and PVA. The characteristic absorption peaks at 1441 and
1272 cm− 1 were attributed to C–O specific angular deformation and
C–H wagging, respectively (Liu, Zhang, Pu, Chen, Li, & Zhong, 2023;
Al-Muntaser, Alamri, Sharma, Eltahir, & Makhlouf, 2022).
Fig. 1 G showed that the XRD pattern of the HPTS/TPB-PVA film was
different from that of the original PVA film. The original PVA film
exhibited four characteristic diffraction peaks at 11.5◦ , 19.5◦ , 22.6◦ , and
40.7◦ , corresponding to indices planes (1 0 0), (1 0 1), (2 0 0), and (1 1 1),
respectively. This indicated that the PVA film possessed a semi­
crystalline structure, which is attributed to the strong intermolecular
hydrogen bonding interaction within PVA chains (Al-Muntaser et al.,
2022). In the XRD curve of HPTS/TPB-PVA film, two new diffraction
peaks at 31.1◦ and 45.5◦ appeared. From this, it can be concluded that
the uniformity of the film was altered, and the structured layout of PVA
was disrupted as a result of the interaction between HPTS, TPB, and
PVA.
3.2. Physical properties of HPTS/TPB-PVA sensing films
The hydrophilic and hydrophobic properties were characterized
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Food Chemistry: X 21 (2024) 101152
based on WCA and WS measurements. The WCA of the PVA films is
21.8◦ . After embedding HPTS/TPB, the WCA of the HPTS/TPB-PVA
sensing film changed to 64.5◦ . This indicated that the hydrophilic
properties of HPTS/TPB-PVA were inferior to those of pure PVA. This
phenomenon may be attributed to the incorporation of TPB molecules,
which changed the cross-linked structure of pure PVA, ultimately
restricting the movement of –OH on the PVA film (Feng, Xu, Shao, Zhu,
Qiu, & Zhu, 2022).
In Table S1, the results presented that the WS of the pure PVA and
HPTS/TPB PVA films were 21.37 % and 3.45 %, respectively. Owing to
the hydrophobicity of the basic component, TPB, in the film, the WS of
the HPTS/TPB PVA film decreased by approximately seven-fold.
Notably, the lower WS of the HPTS/TPB PVA composite film could
prevent the interference caused by water vapor in the package during
the sensing process, which is a good attribute for developing the sensing
tag.
The WVP of pure PVA and HPTS/TPB-PVA were measured to eval­
uate the water molecule diffusion properties of the films. As shown in
Table S1, the WVP values of HPTS/TPB-PVA were lower than those of
pure PVA. The WVP values decreased by 19.85 % (from 0.403 ± 0.05 to
0.323 ± 0.08 g.mm/m2Kpa). This indicates that incorporating the
HPTS/TPB indicator could decrease the WVP of PVA.
From the above results, it can be found that compared to packaging
Q. Han et al.
materials such as starch and chitosan, PVA has excellent physical
properties and is a good choice for the preparation of freshness indicator
labels. This is consistent with the findings of Zhang et al. (Zhang, Sun,
Sang, Jia & Ou, 2022).
3.3. Functional properties of HPTS/TPB-PVA sensing films
Amine vapor, including ammonia, trimethylamine, and triethyl­
amine, is produced during fish degradation (Bondoc, 2016, Bondoc,
2016b). To verify the response of the films to amine vapors, we first
determined the fluorescence color changes of the films exposed to the
headspaces of different amine solutions at the same concentration. As
shown in Fig. 2, the fluorescence signal of the HPTS/TPB-PVA films
gradually changed from pink to yellow, and the color could be detected
with the naked eye. The same trend was observed in the S value ana­
lyses. The S values increased sharply to approximately 30 % before 1.5 h
and then increased gradually from 30 % to 40 % after 1.5 h. It was
inferred that the sensing films exhibited acceptable responses to the
different amine vapors generated during fish storage.
The dynamic color-change process of the HPTS/TPB-PVA sensing
film was recorded for approximately 4 min; the results are shown in
Supplementary Video 1 (20 × speed processing). The sensing film
showed fluorescent color changes within approximately 3 min of drop­
ping 0.01 % ammonia solution onto the surface of the film.
During fish storage, various flavor vapors are generated in the sealed
trays and may interfere with film sensitivity (Martin, Joly, Dupas-
Farrugia, Adt, Oulahal, & Degraeve, 2023). The responses of the films
to several representative flavor vapors were analyzed, and the results are
shown in Fig. 3. As the exposure time of the films to flavor vapors
increased, no pronounced changes in color were observed. The fluo­
rescent color of the sensing films is pink to the naked eye. The trends in
the S values were also analyzed, and no notable changes in the S values
were observed during the entire exposure process. This verified that the
films were good candidates for monitoring fish freshness owing to their
high selectivity.
Fig. 2. (A) Fluorescence changes, and (B) S value trends of the sensing films in ammonia environment.
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Food Chemistry: X 21 (2024) 101152
3.4. Freshness indicators assay
Changes in fish freshness based on freshness indicators were tested
during storage, and the trends of these indicators are presented in Fig. S1
and Fig. S2. As shown in Fig. S1 A and Fig. S2 A, changes in pH values
during the storage (25 ◦ C and 4 ◦ C) showed a similar downward trend at
the beginning, followed by an upward trend. The potential reason for the
decline in pH could be the dissolution of carbon dioxide within the fish
muscle, whereas the increase in pH was likely due to the accumulation of
volatile bases (Chaijan, Benjakul, Visessanguan, & Faustman, 2005).
TBA is a frequently employed indicator of lipid oxidation in fish
muscle (Li, Li, & Hu, 2013). As demonstrated in Figure S1 D and Fig S2
D, there was a progressive increase in the TBA values with increasing
storage time. Because of the absence of clear TBA limit standards in the
aquaculture industry, incorporating other freshness indicators is essen­
tial to assess the freshness of samples comprehensively.
To evaluate the extent of protein decomposition, TVB-N was used as
a freshness indicator (Zhang et al., 2023). In Fig. S1 B, the rate of TVB-N
formation increased gradually before 20 h and then increased rapidly
from 20 to 36 h at the storage temperature of 25 ◦ C. When considering a
level of 25 mg/100 g as the threshold of spoilage (Li, Hu, Li, Zhang, Zhu,
& Li, 2012), TVB-N values reached from the starting value of 6.52 mg/
100 g to 24.85 mg/100 g at 24 h, indicating the shelf life of the fish
sample was 24 h. Correspondingly, in Fig. S2 B, the TVB-N values
increased progressively and reached the upper limit on the 10th day at
4 ◦ C.
As shown in Fig. S1 C, TVC was relatively low in the initial 8 h
(storage at 25 ◦ C) and 4 d (storage at 4 ◦ C), respectively. An apparent
increase was observed during storage. The TVC counts reached 6.56 and
6.38 log CFU/mL at 20 h and on the 8th day, respectively, which were
close to the acceptable limit of 7 log CFU/mL for marine species (Özyurt,
Kuley, Özkütük, Özogul, 2009; Raeisi et al., 2017). These results indi­
cated that the spoilage limit of TVC was slightly shorter than that of
TVB-N during storage.
Based on the above results of these freshness indicators, the
Q. Han et al.
Fig. 3. ΔE value trends of the sensing films in flavor vapors, and the fluorescent images of the films (insert).
threshold of spoilage of the fish sample in this study is 20 h at 25 ◦ C and
8 d at 4 ◦ C.
3.5. Application of the HPTS/TPB-PVA sensing films
The Indicating label was attached to the inner surface of the pack­
aging to monitor the freshness of the samples under two temperature
conditions: 25 and 4 ◦ C. These two levels were chosen for testing
because fish are typically stored and sold under these conditions.
The fish sample was initially monitored for freshness at 25 ◦ C. The
TVB-N and TVC levels in the fish steadily increased with prolonged
storage. Simultaneously, the fluorescence of the HPTS/TPB-PVA film
slowly changed from pink to light pink and finally turned yellow. A
relationship between the TVB-N content and color changes was
observed. As shown in Fig. 4, the TVB-N content was initially low, and
the indicator label appeared pink. After a storage time of 12 h, the TVB-
N content increased to 15.53 mg/100 g. The pink color of the film
gradually faded, indicating that the quality of the fish samples was
within acceptable limits. When the storage time was 24 h, the content of
TVB-N increased to 24.86 mg/100 g, and the film became completely
yellow, indicating that the fish had reached a threshold where it was no
longer suitable for consumption. TVC results indicated that The TVC
content was less than 7 mg/100 g before 20 h, which is below the TVC
standards for edible fish (Zhao, Li, Wang, & Lv, 2012).
Fish spoilage trials were also performed at chilled temperatures
(4 ◦ C). In Fig. 4, the TVB-N at the starting point was 6.52 mg/100 g and
then slowly escalated to 16.61 mg/100 g on the 6th day. It reached
25.62 mg/100 g on the 10th day, exceeding the limit for spoiled fish
(Ruiz-Capillas & Moral, 2001). The fluorescence of the HPTS/TPB-PVA
film changed from dark pink to light pink on the 6th day and then to
yellow after 8 d, indicating that the fish sample spoiled. These results
indicate that the HPTS/TPB-PVA sensing films showed a dramatic color
change to the naked eye at the spoilage point. This proves that the
HPTS/TPB sensing film is a good candidate for intelligent packaging
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Food Chemistry: X 21 (2024) 101152
materials and has the potential for application in the visual monitoring
of fish freshness.
3.6. Packaging test
To verify the fluorescence properties and color changes of the HPTS/
TPB film in a real package, the films were designed and processed as
intelligent sensing tags and attached to commercial packaging of large
yellow croaker fish samples. As shown in Fig. 5 A, a clear fluorescence
color change from pink to yellow was observed through the caps of the
packages when the fish packages were placed under UV light at 365 nm.
A conceptual portable tag reader was also designed for intelligent
monitoring of fish freshness in real time for retailers and consumers. As
shown in Fig. 5 B, the tag reader consists of UV light, a photo window,
and a data processing system that can connect to personal smartphones.
The phone camera was utilized to collect photos of the sensing tag
directly, and detailed data were processed automatically. A portable tag
reader is also an ideal unit for integration with intelligent transportation
vehicles, providing fresh data instantly during transportation.
3.7. Accuracy evaluations
For the intelligent development of the sensing tag technology, the ΔE
values were processed based on the images and fit with the content of
TVB-N. A linear fitting equation can be used to predict the TVB-N levels
during the fish spoiling process quantitatively. As shown in Fig. S3, at
the storage temperature of 25 ◦ C, the linear fitting equation was
y = 4.587x-19.959 within the TVB-N concentrations ranging from 12.7
to 24.8 mg/100 g, with R2 = 0.991. Correspondingly, at the storage
temperature of 4 ◦ C, the calibration equation was described as
y = 4.1712x-12.105, with R2 = 0.990, in the range of 8.4 to 25.6 mg/
100 g.
To validate the accuracy of the predicted results, the TVB-N content
throughout the storage stages was measured using a control method
Q. Han et al. Food Chemistry: X 21 (2024) 101152

Fig. 4. Freshness monitoring of the HPTS/TPB-PVA film at different storage conditions.

Fig. 5. (A) Fluorescence change of HPTS/TPB ratio indicator label in actual samples and (B) the conceptual portable tag reader.

(GB/T 5009.228). The predicted TVB-N content was calculated using the obtained using the developed sensing film and those obtained using the
ΔE values extracted from the images of the sensing film at various stages. control method. When the sensing film exhibited a light pink color, the
Statistical comparison was performed between the predicted results predicted TVB-N values ranged from 14.390 to 15.774 mg/100 g. By

7
Q. Han et al.
contrast, when the sensing film turned yellow, the predicted values of
TVB-N ranged from 24.685 to 25.150 mg/100 g for three different
batches of real samples (see Table S2). Statistical comparisons suggested
no statistically significant differences between these two results, indi­
cating that accurate results could be obtained based on the linear fitting
equation of the color change data. The fabricated HPTS/TPB film can
potentially be used for the visual measurement of fish freshness and
quantitative prediction of TVB-N levels without tedious sample
pretreatment.
4. Conclusions
In this study, ratiometric fluorescent films were successfully devel­
oped for visual monitoring of fish freshness. The pH-sensitive indicator
HPTS was selected as an indicator of amines generated from fish
spoilage, whereas TPB, with AIE properties and highly stable red fluo­
rescence, was selected as an internal control indicator. The HPTS/TPB
ratiometric fluorescent indicator was integrated into the PVA matrix to
obtain HPTS/TPB-PVA films. The films exhibited homogeneous surface
structures. The incorporation of HPTS/TPB reduced the WS and WVP of
the films, which is beneficial for avoiding interference with water vapor
in the package. Under the stimulation of amines released from the fish
samples, the sensing films exhibited distinct fluorescent color changes
from pink to yellow. Furthermore, there is a linear relationship between
the concentration of TVB-N and the ΔE values of the sensing films under
storage conditions of 25 and 4 ◦ C. Therefore, consumers or retailers can
assess the freshness of fish not only visually but also accurately deter­
mine the TVB-N level by analyzing the ΔE values extracted from sensing
film images. This provides a convenient method for analyzing fish
freshness.
CRediT authorship contribution statement
Qian Han: Investigation, Writing – original draft. Min Yang:
Investigation, Writing – original draft. Zexin Zhang: Writing – original
draft, Methodology, Investigation, Conceptualization. Xinwen Bai:
Formal analysis. Xiuying Liu: Writing – original draft, Data curation,
Conceptualization. Zhenhua Qin: Conceptualization, Writing – review
& editing. Wei Zhang: Data curation. Pingping Wang: Writing – review
& editing. Lijie Zhu: Writing – review & editing. Zaixi Shu: Software.
Xuepeng Li: Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgements
This research was funded by the National Key R&D Program of China
(2019YFD0901605), and the Scientific Research Foundation of Wuhan
Polytechnic University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fochx.2024.101152.
8
Food Chemistry: X 21 (2024) 101152
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